Seo Hwa-Seon, Serra Rosa
Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA.
Dev Biol. 2007 Oct 15;310(2):304-16. doi: 10.1016/j.ydbio.2007.07.040. Epub 2007 Aug 9.
In this study, we address the function of Transforming Growth Factor beta (TGF-beta) and its type II receptor (Tgfbr2) in limb development in vivo. Mouse embryos were generated in which the Tgfbr2 gene was deleted in early limb mesenchyme using Prx1Cre-mediated LoxP recombination. A high level of Tgfbr2 gene deletion was verified in limb mesenchyme by PCR between E9.5 and E10.5 days in Cre expressing mice. RT-PCR assays indicated a significant depletion of Tgfbr2 mRNA by E10.5 days as a result of Cre mediated gene deletion. Furthermore, limb mesenchyme from Cre(+);Tgfbr2(f/f) mice placed in micromass culture did not respond to exogenously added TGF-beta1 confirming the functional deletion of the receptor. However, there was an unexpected increase in the number and intensity of Alcian blue stained chondrogenic nodules in micromass cultures derived from Tgfbr2-deleted limbs relative to cultures from control limbs suggesting that Tgfbr2 normally limits chondrogenesis in vitro. In vivo, early limb development and chondrocyte differentiation occurred normally in Tgfbr2-depleted mice. Later in development, depletion of Tgfbr2 in limb mesenchyme resulted in short limbs and fusion of the joints in the phalanges. Alteration in the length of the long bones was primarily due to a decrease in chondrocyte proliferation after E13.5 days. In addition, the transition from prehypertrophic to hypertrophic cells was accelerated while there was a delay in late hypertrophic differentiation leading to a reduction in the length of the marrow cavity. In the joint, cartilage cells replaced interzone cells during development. Analysis of markers for joint development indicated that the joint was specified properly and that the interzone cells were initially formed but not maintained. The results suggest that Tgfbr2 is required for normal development of the skeleton and that Tgfbr2 can act to limit chondrogenesis in mesenchymal cells like the interzone.
在本研究中，我们探讨了转化生长因子β（TGF-β）及其II型受体（Tgfbr2）在体内肢体发育中的功能。利用Prx1Cre介导的LoxP重组技术构建了在早期肢体间充质中Tgfbr2基因被敲除的小鼠胚胎。通过PCR在E9.5至E10.5天对表达Cre的小鼠肢体间充质中的Tgfbr2基因敲除水平进行了验证。RT-PCR分析表明，由于Cre介导的基因敲除，到E10.5天时Tgfbr2 mRNA显著减少。此外，来自Cre(+)；Tgfbr2(f/f)小鼠的肢体间充质在微团培养中对外源添加的TGF-β1无反应，证实了受体的功能缺失。然而，与对照肢体培养物相比，来自Tgfbr2基因敲除肢体的微团培养物中阿尔新蓝染色的软骨结节数量和强度意外增加，这表明Tgfbr2通常在体外限制软骨生成。在体内，Tgfbr2基因敲除小鼠的早期肢体发育和软骨细胞分化正常。在发育后期，肢体间充质中Tgfbr2的缺失导致肢体短小和指骨关节融合。长骨长度的改变主要是由于E13.5天后软骨细胞增殖减少。此外，从增殖前软骨细胞向肥大软骨细胞的转变加速，而晚期肥大分化延迟，导致骨髓腔长度缩短。在关节中，软骨细胞在发育过程中取代了中间区细胞。关节发育标志物分析表明，关节指定正常，中间区细胞最初形成但未维持。结果表明，Tgfbr2是骨骼正常发育所必需的，并且Tgfbr2可以像中间区一样在间充质细胞中限制软骨生成。
